Detailed knowledge of the basic events underlying insulin's ability to interact with cells will lead to a better understanding of the mechanisms of insulin action and of disease states in which insulin action is abnormal. Therefore, the overall goals of this research proposal are to elucidate the molecular and biochemical events which occur after insulin binds to its cell surface receptor. (1) Over the past three years we have conducted a number of studies with a kinase defective (AK1018) insulin receptor. This receptor inhibits the function of the normal native receptors in the host rat fibroblasts producing cellular insulin resistance. We advanced the substrate competition hypothesis to explain this phenomenon and propose to further explore these findings by constructing new receptor mutants which should cause inhibition of normal receptor function. We will attempt to directly identify substrates by cross linking and other methodologies using mutant insulin receptors and specific cytoplasmic domains. (2) Our recent studies of the truncated insulin receptor mutant (CT) have shown that this receptor has normal kinase activity, signals insulin's metabolic effects poorly, but has a super normal ability to transmit mitogenic signalling. We will test the idea of specific functional domains, with respect to biologic signalling, by creating deletion insulin receptor mutants in attempts to generate pure "metabolic" and pure "mitogenic" insulin receptors. (3) We and other shave shown divergent as well as convergent signalling mechanisms for the heterologous insulin and IGF-I receptors. We plan to assess the mechanism of this further by studying specific domain functions of these two receptors. This will be done by cresting chimeric insulin:IGF-I receptors in which the c-tails of these receptors will be swapped. (4) Cytoplasmic receptor sequences necessary for endocytosis will be identified as well as ectodomain regions necessary for biologic signalling. (5) We will continue our studies of the cellular pathway, or itinerary, of insulin and its receptor following the initial binding event. These studies will involve the use of normal cell lines as well as cells transfected with mutated insulin receptors. An EM immunohistochemical approach will also be utilized to study subcellular localization of normal and mutated insulin receptors. The studies outlined in this proposal will apply new molecular and biochemical techniques to provide mechanistic information which will further our understanding of the cellular actions of this important hormone.